Reversed phase high performance liquid chromatography (RP-HPLC) is used ubiquitously in academic institutions, forensic laboratories, fine chemicals, and pharmaceutical industries etc. for the analysis, characterization, separation, purification and/or isolation of small organic molecules, natural products, and biologically active molecules such as polypeptides, proteins, and nucleotides. In the pharmaceutical industry, analytical RP-HPLC is used for the release and characterization of raw materials, intermediates, and active pharmaceutical ingredients (APIs). Preparative reversed phase high performance liquid chromatography (Prep-RP-HPLC) is used for the commercial production of Peptide APIs, and most other complex APIs that are not amenable to crystallization.
Preparative RP-HPLC in the elution mode is limited by the loading capacity of the analyte. In the elution preparative RP-HPLC mode, the typical loading capacity of synthetic peptides is in the range of 1 to 2 mgs per ml of packed column volume (viz., 0.1% to 0.2% with respect to total column volume).
The patent application US20120322976 discloses a preparative HPLC of a GLP-1 analog. The loading was 0.225% with respect to total column volume {(about 45 mgs on to a 20 ml C-18 substituted (Octadecyldimethylsilyl) silica resin (particle size: 15 microns)}.
The patent application US20110313131 discloses a preparative HPLC of (Aib 8, 35) GLP-1(7-36)-NH2 at loadings up to 20 g/L (2% with respect to total column volume).
Recent advances in RP-HPLC have focussed on producing spherical silica and development of new bonding chemistries to furnish stationary supports that have improved stability and selectivity. The earlier supports were irregular silica particles that were derivatized with C-18 or C-8 chains, and they suffered from high back pressure. The high back pressure limited their use with respect to quantity that could be purified in a single run, and to relatively smaller diameter columns.
The commercial manufacture of spherical silica that has been derivatized by C-18, C-8, and other ligands has overcome these challenges and has extended the utility of preparative HPLC vastly. These technological advances in the bonded silica supports and process HPLC instrumentation have made possible commercial production of complex peptides such as Fuzeon®, a 36-amino acid peptide, in ton quantities. Unfortunately, these large scale HPLC instruments and the associated column hardware are very costly and restrict the affordability of the methods.
Further, RP-HPLC in the displacement mode has better loading capacity than RP-HPLC in the elution mode but it is arduous to develop. The displacement chromatography is best suited for ion exchange mode, and has found numerous recent applications.
Displacement chromatography utilizes as mobile phase a displacer solution which has higher affinity for the stationary phase material than do the sample components. The key operational feature which distinguishes displacement chromatography from elution chromatography is the use of a displacer molecule.
The U.S. Pat. No. 6,239,262 discloses low molecular weight displacers for protein purification in hydrophobic interaction and reverse phase chromatographic systems.
In displacement chromatography separations, the sample components are introduced in the form of homogeneous sample solution, so that individual components are each delivered at a constant concentration throughout the sample application step. The driving force for separation is that weak binders are displaced from the limited number of binding sites on the stationary phase material by more strongly binding components of the product mixture. This proceeds in a continuous manner until the product and other stronger binders are fully retarded in the earlier part of the chromatography bed, thus permitting the more weakly binding impurities to stay bound to the stationary phase material further along the chromatography bed. Once all sample molecules are bound to the stationary phase, no further movement of these molecules will be observed. A problem which may occur because of such use of homogeneous sample solutions, however, is that molecules of strongly binding components introduced during an early part of sample application may inadvertently be displaced by weaker binders introduced during a later stage of sample application.
Therefore there is a need for a simple, cost effective and scalable Prep-RP-HPLC process for peptides.